Glucocorticoids

ABSTRACT

PCT No. PCT/EP94/00937 Sec. 371 Date Oct. 6, 1995 Sec. 102(e) Date Oct. 6, 1995 PCT Filed Mar. 24, 1994 PCT Pub. No. WO94/22898 PCT Pub. Date Oct. 13, 1994Glucocorticoids of general formula IR-Val-O-GC (II),are described, in which O-GC is the radical of a 21-hydroxycorticoid that has an antiinflammatory action, Val represents a valine radical in the 21-position of the corticoid and R means a hydrogen atom or a hydrocarbon radical with up to 32 carbon atoms that is optionally substituted by hydroxy groups, amino groups, oxo groups and/or halogen atoms and/or interrupted by oxygen atoms, SO2 groups and/or NH groups and their salts.

This application is a 371 of PCT/EP94/00937 filed Mar. 24, 1994.

This application is a 371 of PCT/EP94/00937 filed Mar. 24, 1994.

The invention relates to glucocorticoids of general formula I

    R--Val--O--GC (I),

in which

O-GC is the radical of a 21-hydroxycorticoid that has anantiinflammatory action,

Val represents a valine radical in the 21-position of the corticoid, and

R means a hydrogen atom or a hydrocarbon radical with up to 32 carbonatoms that is optionally substituted by hydroxy groups, amino groups,oxo groups and/or halogen atoms and/or interrupted by oxygen atoms, SO₂groups and/or NH groups and their salts.

The glucocorticoids according to the invention are valuable intermediateproducts and/or pharmacologically effective substances which are used,i.a., for the treatment of inflammatory conditions that arecharacterized by increased activity of the enzyme leukocyte-elastase.

Glucocorticoids are the best-known class of antiinflammatory activeingredients. Owing to their broad range of uses and their greatantiinflammatory action, corticoid preparations are therapeutic agentsof first choice in a wide variety of inflammatory diseases, such as, forexample, diseases of the rheumatoid group, allergies, inflammatorydiseases of the lungs, heart, and intestines, bronchial asthma,hyperproliferative diseases of the skin (psoriasis), eczemas,auto-immune diseases, or states of shock.

Their potential side effects, such as suppression of the brain-pituitarygland-suprarenal axis, their catabolic action, their influence on saltand water balance, osteoporosis, their influence on the blood-sugarlevel in the case of diabetics or the induction of skin atrophy in thecase of topical application keep this class of substances from being putto an even broader range of therapeutic uses. According to presentknowledge, these side effects, just like the antiinflammatory action ofthe glucocorticoids, are mediated by the same receptor. Up to now,therefore, reductions of the side-effect potential have been achieved byincreasing the metabolic clearance of local-action corticoids (anti-drugprinciple). More lipophilic prodrugs are supposed to promote thepenetration of corticoids into the skin and improve the retention ofcorticoids in the lungs. Despite the reduced side-effect potential ofmodern corticoids, especially long-term treatment with activeingredients of this class of substances remains critical.

Leukocyte-elastase is a serine-protease with a molecular weight of about30,000 D. It is formed in promyelocytes and is found mainly inneutrophilic granulocytes [Duswald, K. H. (1983), Zur Pathobiochemie derLeukozyten-Elastase [Pathobiochemistry of Leukocyte-Elastase], GITVerlag Ernst Giebler, Darmstadt.] Their occurrence is also described inMonozyten, Lymphozyten und eosinophilen Granulozyten [Monocytes,Lymphocytes and Eosinophilic Granulocytes] [Kargi et al. (1990)].Elastase and Cathepsin G of Human Monocytes: Heterogeneity andSubcellular Localization to Peroxidase-Positive Granules. The Journal ofHistochemistry and Cytochemistry 38: 1179-1186; Lungarella et al.(1992). Identification of Elastase in Human Eosinophils:Immunocalization, Isolation and Partial Characterization. Archives ofBiochemistry and Biophysics, 292: 128-135; Bristow et al. (1991).Elastase is a constituent product of T cells. Biochemical andBiophysical Research Communications, 181: 232-239.] The enzyme issecreted in vitro after stimulation of granulocytes and monocytes[Schmidt (1978). Differential Release of Elastase and Chymotrypsin fromPolymorphonuclear Leukocytes. In: Neutral Proteases of HumanPolymorphonuclear Leukocytes. Havemann and Janoff (Editors) Urban &Schistzenberg, Inc. Baltimore, Munich; Xie et al. (1993). Release ofElastase from Monocytes Adherent to a Fibronectin-Gelatin Surface. Blood81: 186-192.] High elastase activities are observed in vivo in the caseof inflammatory diseases of the lungs, in the case of rhinitis, insynovial fluid in the case of rheumatoid arthritis, and on the skinsurface in the case of different forms of eczema [Tanaka et al. (1990).A Sensitive and Specific Assay for Granulocyte Elastase in InflammatoryTissue Fluid Using L-Pyroglutamyl-L-prolyl-L-valine-p-nitroanilide.Clinica Chimica Acta 187: 173-180; Wiedow et al. (1992). LesionalElastase Activity in Psoriasis, Contact Dermatitis, and AtopicDermatitis. Journal for Investigative Dermatology 99: 306-309.].Elastase inhibitors are developed as therapeutic agents for theindication of pulmonary emphysema. Leukocyte-elastase cleaves ester andpeptide bonds C-terminally of a short recognition sequence. Therecognition sequence is readily characterized. The C-terminal radicalplays a small role with regard to the catalytic activity of the enzyme[Castillo et al. (1979). Sensitive Substrates for Human Leukocyte andPorcine Pancreatic Elastase: A Study of the Merits of VariousChromophoric and Fluorogenic Leaving Groups In Assays for SerineProteases. Analytical Biochemistry 99:53-64.]. The attempt to achieve anantiinflammatory action by inhibiting leukocyte-elastase is known.

It is also known that elastase transcortin cleaves the transport proteinfrom physiological corticoids that have only a weak antiinflammatoryaction (hydrocortisone, corticosterone). The cleavage product of thisprotein has a significantly reduced affinity for the glucocorticoid. Thehypothesis is based on the idea that an inflammation-specific release ofcorticoids could be produced by the activity of leukocyte-elastase[Hammond et al. (1990). A Role for Corticosteroid-Binding Globulin inDelivery of Cortisol to Activated Neutrophile. Journal of ClinicalEndocrinology and Metabolism 71: 34-39; Hammond et al. (1990).Interaction Between Corticosteroid Binding Globulin and ActivatedLeukocytes in Vitro. Biochemical and Biophysical Research Communications172: 172-177.]. The idea of using the enzymatic activity ofleukocyte-elastase for inflammation-specific activation of inactivecorticoid prodrugs is new. Such compounds could reduce the side-effectpotential of glucocorticoids, e.g., in dermatological indications,diseases of the rheumatoid group or with use of glucocorticoids ininflammatory lung diseases.

The glucocorticoid prodrugs are cleaved into the effectiveglucocorticoid and the pharmacologically inactive peptide by thecatalytic activity of leukocyte-elastase. In this way, the activeglucocorticoid is released selectively at the focus of inflammation.This circumstance results in an increase of the concentration of theactive glucocorticoid in the focus of inflammation. The concentrationsof the active glucocorticoid in non-inflamed areas are thus kept to aminimum. As a result, a reduction in the local and systemic side-effectpotential is achieved.

Glucocorticoid prodrugs according to the invention that can be used are,for example, those which are derivatives of 21-hydroxycorticoids, whichare mentioned in EPA 0098 566 or the "Red List" [Herausg. Bundesverbandder Pharmazeutischen Chemie e.V. [Editors Registered Association ofPharmaceutical Chemistry] Frankfurt and Main]. As radicals R of theprodrugs, for example, acyl radicals with 1 to 12 carbon atoms, benzylradicals or those which, with the valine radical, form an oligopeptideradical with 2 to 6 amino acids, optionally provided with the usualprotective groups, are considered.

Preferred oligopeptide radicals are those of aliphatic amino acids andespecially those which contain alanine, proline, and valine as aminoacids.

Suitable protective groups are, for example, those which are listed involume XV/1 of "Methoden der Organischen Chemie [Methods of OrganicChemistry]" [Houben-Weyl, p. 20 ff].

Especially preferred glucocorticoids are those of general formula IIaccording to the invention ##STR1## in which R¹ =H, CH=O (C=O)R",(C=O)OR" or SO₂ R"

X¹ --X³ =independently of one another, alanine, proline or valine,

A-B=CH₂ --CH₂ or CH=CH

R⁶ =H, F, Cl, Me,

R⁹ =H, F, Cl,

R¹⁶ =H, Me, OH,

R¹⁷ =H, OH, O(C=O)R'" or

R¹⁶, R¹⁷ =is alkylidenedioxy, in which

R" represents a hydrocarbon radical that contains C₁ -C₁₈ and

R'" represents a C₁ -C₁₀ alkyl (straight-chain or branched-chain) aryl,alkylaryl, or C₁ -C₃ alkoxy radical and

the alkylidene radical is derived from an aliphatic aldehyde thatcontains 1-6 C atoms, a ketone that contains 3-6 C atoms, or a cyclicketone or benzaldehyde that contains 5-6 C atoms and their salts.

The preferred recognition sequence consists of X¹ -X³ =independently ofone another, alanine, proline or valine and especially of X¹, X²=alanine, X³ =proline, valine. Any other tetrapeptide sequence, however,can also be used within the scope of the invention, if it bonds toelastase and is accepted as substrate.

Suitable hydrocarbon radicals R" and R'" are those which are derivedfrom the already-mentioned protective groups.

The synthesis of the glucocorticoids according to the invention is doneaccording to the processes that are familiar to one skilled in the art,as they are used in general in the case of peptide syntheses ("Methodender Organischen Chemie" (Houben-Weyl, Vol. XV/1 and XV/2). The sampleapplications below are used to explain the invention in greater detail.

The abbreviations that are used are:

Aib=aminoisobutyric acid, Ala=alanine, Gly=glycine, Pro=proline,Val=valine; BMV=betamethasone-17-valerate, FC =fluocortolone,HC=hydrocortisone, MP=6α-methylprednisolone,MPP=6α-methylprednisolone-17-propionate, TCA=triamcinolone acetonide;Ac=acetyl, ASA=amino acid analysis, Boc=tertbutoxycarbonyl, Bz=benzoyl,Cbs=4-chlorobenzenesulfonyl, Cbp =3-(9-carbazolyl)-propionic acid,DDC=dicyclohexylcarbodiimide, DMAP=4-(N,N-dimethylamino)-pyridine,DPAc=diphenylacetyl, DPC=diphenylcarbamoyl,Fmoc=9-fluorenylmethoxycarbonyl, NCA=N-carbonic anhydrides,Piv=pivaloyl, Pht, phthyloyl, TFA=trifluoroacetic acid,Z=benzyloxycarbonyl.

The IUPAC recommendations for the naming of peptides [Biochem. J. 219,345 (1984)] are observed in these sample applications.

EXAMPLES OF SYNTHESIS EXAMPLE 1

N-(1,1-Dimethylethoxycarbonyl)-valine[11β,21-dihydroxy-3,20-dioxo-6α-methyl-17-propionyloxy-pregna-1,4-dien-21-yl]ester (Boc-Val-O-MPP)

A solution of 2.15 g (5.00 mmol) of 6α-methylprednisolone-17-propionatein 25 ml of dichloromethane is mixed with 1.36 g (6.25 mmol) ofN-(1,1-dimethylethoxycarbonyl)-valine and 121 mg (0.99 mmol) of4-dimethylaminopyridine. Then, a solution of 1.36 g (6.59 mmol) ofdicyclohexylcarbodiimide in 5 ml of dichloromethane is added in oneshot. After 1-3 hours, the precipitate that is produced is suctioned offand washed with diethyl ether. The filtrate is concentrated byevaporation in a vacuum. Chromatography on silica gel(hexane→hexane/ethyl acetate 1:1) provides 3.01 g (96%) ofN-(1,1-dimethylethoxycarbonyl)-valine [11β,21-dihydroxy-3,20-dioxo-6α-methyl-17-propionyloxy-pregna-1,4-dien-21-yl]ester. Crystallization from dichloromethane/diisopropyl ether. Meltingpoint 110°-130° C. [α]_(D) =+32° (chloroform).

Example 2

Valine[11β,21-dihydroxy-3,20-dioxo-6α-methyl-17-propionyloxy-pregna-1,4-dien-21-yl]ester trifluoroacetate (H-Val-O-MPP-TFA)

Under initial cooling, 5 ml of trifluoroacetic acid is poured over 896mg (1.42 mmol) of N-(1,1-dimethylethoxycarbonyl)valine[11β,21-dihydroxy-3,20-dioxo-6α-methyl-17-propionyloxy-pregna-1,4-dien-21-yl]ester, and it is stirred for 1-2 hours at room temperature. Then, thetrifluoroacetic acid is evaporated in a vacuum. The residue is taken upin a little dichloromethane, 5 ml of toluene is added and thenevaporated to dryness in a vacuum. The residue is crystallized. 728 mg(80%) of valine [11β,21-dihydroxy-3,20-dioxo-6α-methyl-17-propionyloxy-pregna-1,4-dien-21-yl] ester trifluoroacetate is obtained. Meltingpoint 148° C.

Example 3

N-(N-(N-(N-(1,1-Dimethylethoxycarbonyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,21-dihydroxy-3,20-dioxo-6α-methyl-17-propionyloxy-pregna-1,4-dien-21-yl]ester (Boc-Ala-Ala-Pro-Val-O-MPP) (Boc-Ala-Ala-Pro-Val-O-MPP)

336 mg (0.52 mmol) of valine [11β,21-dihydroxy-3,21-dioxo-6α-methyl-17-propionyloxy-pregna-1,4-dien-21-yl] estertrifluoroacetate, 180 mg (0.50 mmol) ofN-(1,1-dimethylethoxycarbonyl)-alanyl-alanyl-proline, 68 mg (0.50 mmol)of hydroxybenzotriazole are dissolved in 20 ml of dichloromethane, and150 mg (0.72 mmol) of dicyclohexylcarbodiimide in 2 ml ofdichloromethane is added. Then, 55 ml (0.50 mmol) of N-methylmorpholineis immediately added and stirred for 2 hours at room temperature. Forworking-up, it is filtered off from dicyclohexylurea, rewashed withdiethyl ether and the combined organic phases are washed with 40 ml of0.5N HCl, 0.5N NaOH and saturated sodium chloride solution in each caseand dried on sodium sulfate. Evaporation of the solvent in a vacuumyields the crude product. Chromatography on silica gel(hexane→hexane/acetone 1:1) yields 402 mg (89%) ofN-(N-(N-(N-(1,1-dimethylethoxycarbonyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,21-dihydroxy-3,20-dioxo-6α-methyl-17-propionyloxy-pregna-1,4-dien-21-yl]ester, crystallization from dichloromethane/diisopropyl ether/hexane.

Melting point sintering starting from 135° C., decomposition startingfrom 160° C., [α]_(D) =-28° (c=0.5% in chloroform), HPLC: 99%, ASA: Ala20.4 Pro 0.95 Val 1.01, racemic test (GC): D-Ala<1% D-Pro<1% D-Val<1%.

Example 4

N-(N-(N-(N-(9H-Fluoren-9-ylmethoxycarbonyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,21-dihydroxy-3,20-dioxo-6α-methyl-17-propionyloxy-pregna-1,4-dien-21-yl]ester (Fmoc-Ala-Ala-Pro-Val -O-MPP)

As described in Example 3, 631 mg (0.98 mmol) of valine[11β,21-dihydroxy-3,21-dioxo-6α-methyl-17-propionyloxy-pregna-1,4-dien-21-yl] ester trifluoroacetate, 446 mg (0.93 mmol) ofN-(9H-fluoren-9-ylmethoxycarbonyl)-alanyl-alanyl-proline, 136 mg (1.00mmol) of hydroxybenzotriazole, 215 mg (1.04 mmol) ofdicyclohexylcarbodiimide and 220 ml (2.00 mmol) of N-methylmorpholineare reacted. Chromatography on silica gel(dichloromethane→dichloromethane/methanol 97:3) yields 400 mg (42%) ofN-(N-(N-(N-(9H-fluoren-9-ylmethoxycarbonyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,21-dihydroxy-3,20-dioxo-6α-methyl-17-propionyloxy-pregna-1,4-dien-21-yl] ester. Crystallization from dichloromethane/diisopropyl ether/hexane.

[α]_(D) =-24° (c=0.5% in chloroform), HPLC: 98%.

Racemic test (GC): D-Ale<1% D-Pro<1% D-Val<1%

Example 5

N-(N-(N-(N-(Phenylmethoxycarbonyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine [11β21-dihydroxy-3,20-dioxo-6α-methyl-17-propionyloxy-pregna-1,4-dien-21-yl] ester (Z-Ala-Ala-Pro-Val-O-MPP)

Under the conditions indicated in Example 3, 131 mg (0.20 mmol) ofvaline[11β,21-dihydroxy-3,21-dioxo-6α-methyl-17-propionyloxy-pregna-1,4-dien-21-yl]ester trifluoroacetate, 72 mg (0.19 mmol) ofN-(benzyloxycarbonyl)-alanyl-alanyl-proline (Z-Ala-Ala-Pro-OH), 51 mg(0.37 mmol) of hydroxybenzotriazole, 46 mg (0.22 mmol) ofdicyclohexylcarbodiimide and 30 ml (0.28 mmol) of N-methylmorpholine arereacted. Gradient chromatography on silica gel(dichloromethane→dichloromethane/methanol 9:1) provides 101 mg (60%) ofN-(N-(N-(N-(phenylmethoxycarbonyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,21-dihydroxy-3,20-dioxo-6α-methyl-17-propionyloxy-pregna-1,4-dien-21-yl]ester.

Crystallization from methanol/diisopropyl ether.

Melting point starting from 137° C. (dec.), [α]_(D) =-23° (c=0.5% inchloroform), HPLC: 98-99%.

Racemic test (GC): D-Ala 1.8% D-Pro<1% D-Val<1%.

Example 6

N-(N-(N-(L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,21-dihydroxy-3,20-dioxo-6α-methyl-17-propionyloxy-pregna-1,4-dien-21-yl]ester trifluoroacetate (H-Ala-Ala-Pro-Val-O-MPP-TFA)

3.5 g (4.0 mmol) of Boc-Ala-Ala-Pro-Val-O-MPP (Example 3) is dissolvedin 50 ml of dichloromethane and mixed with 6.5 ml of trifluoroaceticacid at 0° C. After 2.5 hours, the solvent is removed i.vac. (5 HPA),the residue is taken up in dichloromethane and precipitated withdiisopropyl ether. 3.5 g (99%) ofN-(N-(N-(L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,21-dihydroxy-3,20-dioxo-6α-methyl-17 -propionyloxy-pregna-1,4-dien-21-yl] ester trifluoroacetate is obtained.

Melting point 164°-178° C. (dec), [α]_(D) =-19° (c=0.5% in chloroform) ,HPLC: 98%

ASA: Ala 1.93 Pro 1.09 Val 0.98, racemic test (GC): D-Ala <1% D-Pro<1%D-Val<1%.

Example 7

N-(N-(N-(N-(Methylcarbonyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,21-dihydroxy-3,20-dioxo-6α-methyl-17-propionyloxy-pregna-1,4-dien-21-yl] ester (Ac-Ala-Ala-Pro-Val-O-MPP)

500 mg (0.57 mmol) of H-Ala-Ala-Pro-Val-O-MPP x TFA (Example 3) isdissolved in 20 ml of dichloromethane, mixed with 115 μl (1.21 mmol) ofacetic anhydride and stirred for 24 hours at room temperature. Theresidue that remains after removal of the solvent i.vac. ischromatographed (200 g of silica gel 60,dichloromethane→dichloromethane/methanol 9:1) 344 mg (75%) ofN-(N-(N-(N-(methylcarbonyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,21-dihydroxy-3,20-dioxo-6α-methyl-17-propionyloxy-pregna-1,4-dien-21-yl] ester, and, after recrystallization, 305 mg(66%) in crystalline form from methanol/diisopropyl ether/hexane areobtained.

Melting point 152°-155° C. sintering: 174° C., [α]_(D) =-28° (c=0.5% inchloroform), HPLC: 98-99%.

Racemic test (GC): D-Ala 2.3% D-Pro<1% D-Val<1%.

Example 8

N-(N-(N-(N-(Phenylcarbonyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,21-dihydroxy-3,2 0-dioxo-6α-methyl-17-propionyloxy-pregna-1,4-dien-21-yl] ester (Bz-Ala-Ala-Pro-Val-O-MPP)

250 mg (0.28 mmol) of H-Ala-Ala-Pro-Val-O-MPP x TFA (Example 3) isdissolved in 50 ml of dichloromethane and 5 ml of triethylamine, mixedwith 49 μl (0.37 mmol) of benzoyl chloride and stirred for 28 hours atroom temperature. For working-up, it is washed with water and saturatedsodium chloride solution, dried on sodium sulfate and concentrated byevaporation i.vac. The residue that remains after removal of the solventi.vac. is chromatographed. Gradient chromatography on 200 g of silicagel 60 (acetone/hexane 1:2→2:1) yields 153 mg (62%) ofN-(N-(N-(N-(phenylcarbonyl) -L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,21-dihydroxy-3,20-dioxo-6α-methyl-17-propionyloxy-pregna-1,4-dien-21-yl] ester, and, after recrystallization, 131 mg (53%) in crystallineform from dichloromethane/diisopropyl ether.

Melting point starting from 150° C. decomposition [α]_(D) =-14° (c=0.5%in chloroform), HPLC: 95-98%.

Racemic test (GC): D-Ala 5.0% D-Pro<1% D-Val<1%.

Example 9

N-(N-(N-(N-(Valeroyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,21-dihydroxy-3,20-dioxo-6α-methyl-17-propionyloxy-pregna-1,4-dien-21-yl] ester (Valeroyl-Ala-Ala-Pro-Val-O-MPP)

307 mg (0.35 mmol) of H-Ala-Ala-Pro-Val-O-MPP x TFA (Example 3), 36 μl(0.33 mmol) of valeric acid, 52 mg (0.35 mmol) ofN-hydroxy-benzotriazole and 74 μl (0.66 mmol) of N-methylmorpholine aredissolved in 40 ml of dichloromethane, mixed with a solution of 84 mg(0.41 mmol) of dicyclohexylcarbodiimide in 10 ml of dichloromethane andstirred for 4 hours at room temperature. For working-up, theprecipitated urea is filtered off, the filtrate is diluted with 40 ml ofdichloromethane and washed with 40 ml each of 0.5N NaOH, 0.5N HCl andsaturated sodium chloride solution, dried on sodium sulfate andconcentrated by evaporation i.vac. The residue that remains afterremoval of the solvent i.vac. is chromatographed. Gradientchromatography on 200 g of silica gel 60(dichloromethane→dichloromethane/methanol 95:5) yields 180 mg (64%) ofN-(N-(N-(N -(valeroyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,21-dihydroxy-3,20-dioxo-6α-methyl-17-propionyloxy-pregna-1,4-dien-21-yl] ester and, after recrystallization, 157 mg (56%) in crystallineform from dichloromethane/diisopropyl ether/hexane.

Melting point 142° C., [α]_(D) =-54° (c=0.5% in methanol), HPLC: 94-98%.

Racemic test (GC): D-Ala 1.7% D-Pro<1% D-Val<1%.

Example 10

N-(N-(N-(N-((Diphenylmethyl)carbonyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,21-dihydroxy-3,20-dioxo-6α-methyl-17-propionyloxy-pregna-1,4-dien-21-yl]ester (DPAc-Ala-Ala-Pro-Val -O-MPP)

150 mg (0.17 mmol) of H-Ala-Ala-Pro-Val-O-MPP x TFA (Example 3), 40 mg(0.19 mmol) of diphenylacetic acid, 23 mg (0.17 mmol) ofN-hydroxy-benzotriazole and 41 μl (0.66 mmol) of diisopropylethylamineare dissolved in 3 ml of dichloromethane, mixed with a solution of 69 mg(0.33 mmol) of dicyclohexylcarbodiimide in 4 ml of dichloromethane andstirred for 23 hours at room temperature. For working-up, theprecipitated urea is filtered off and the residue that remains afterremoval of the solvent i.vac. is chromatographed (gradientchromatography, 24 g of silica gel 60, hexane→hexane/acetone 1:2). 148mg (91%) of N-(N-(N-(N-((diphenylmethyl)carbonyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,21-dihydroxy-3,20-dioxo-6α-methyl-17-propionyloxy-pregna-1,4-dien-21-yl]ester is obtained, which is purified again on a Lichroprep Si60Aprepacked column (hexane→hexane/dichloromethane/methanol 30:60:10) (86mg ₋₋ 53%) and 75 mg (46%) of crystalline compound fromdichloromethane/diisopropyl ether is obtained.

Melting point sintered starting from 157° C., 177° C., HPLC: 99%.

ASA: Ala 1.90 Pro 1.10 Val 1.00, racemic test (GC): D-Ala 2.9% D-Pro2.1% D-Val 0.8%.

Example 11

N-(N-(N-(N-(Diphenylcarbamoyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,21-dihydroxy-3,20-dioxo-6α-methyl-17-propionyloxy-pregna-1,4-dien-21-yl] ester (DPC-Ala-Ala-Pro-Val-O-MPP)

150 mg (0.17 mmol) of H-Ala-Ala-Pro-Val-O-MPP x TFA (Example 3) and 150μl (0.89 mmol) of diisopropylethylamine are dissolved in 6 ml ofdichloromethane, mixed with 155 mg (0.37 mmol) of diphenylcarbamoylchloride and stirred for 110 hours at room temperature. The residue thatremains after removal of the solvent i.vac. is chromatographed. Gradientchromatography (Lichroprep Si60A pre-packed column,hexane→hexane/dichloromethane/methanol 30:60:10) yields 158 mg (97%) ofN-(N -(N-(N-(diphenylcarbamoyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,21-dihydroxy-3,20-dioxo-6α-methyl-17-propionyloxy-pregna-1,4-dien-21-yl] ester, and after recrystallization, 112 mg (68%) incrystalline form from dichloromethane/diisopropyl ether.

Melting point 150°-152° C., HPLC: 95.6-98.2%, racemic test (GC): D-Ala2.9% D-Pro 2.0% D-Val<1%.

Example 12

N-(N-(N-(N-(2-Naphthoyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,21-dihydroxy-3,20-dioxo-6α-methyl-17-propionyloxy-pregna-1,4-dien-21-yl] ester (2-Naphthoyl-Ala-Ala-Pro-Val-O-MPP)

120 mg (0.14 mmol) of H-Ala-Ala-Pro-Val-O-MPP x TFA (Example 3), 28 mg(0.16 mmol) of 2-naphthoic acid, 19 mg (0.14 mmol) ofN-hydroxy-benzotriazole and 100 μl (0.57 mmol) of diisopropylethylamineare dissolved in 3 ml of dichloromethane, mixed with a solution of 67 mg(0.33 mmol) of dicyclohexylcarbodiimide in 1 ml of dichloromethane andstirred for 18 hours at room temperature. For working-up, theprecipitated urea is filtered off and the residue that remains afterremoval of the solvent i.vac. is chromatographed (gradientchromatography on 23 g of silica gel 60, acetone/hexane 1:1→2:3). 103 mg(82%) of N-(N-(N-(N-(2-naphthoyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,21-dihydroxy-3,20-dioxo-6α-methyl-17-propionyloxy-pregna-1,4-dien-21-yl]ester and, after recrystallization, 87 mg (69%) in crystalline form fromdichloromethane/diisopropyl ether are obtained.

Melting point 166°-170° C., HPLC: 97.6-97.9%, ASA: Ala 1.90 Pro 1.12 Val0.98, racemic test (GC): D-Ala 7.3% D-Pro 2.0% D-Val 0.8%.

Example 13

N-(N-(N-(N-(3-Phenyl-butenoyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,21-dihydroxy-3,20-dioxo-6α-methyl-17-propionyloxy-pregna-1,4-dien-21-yl] ester (Cinnamoyl-Ala-Ala-Pro-Val-O-MPP)

120 mg (0.14 mmol) of H-Ala-Ala-Pro-Val-O-MPP x TFA (Example 3), 24 mg(0.16 mmol) of cinnamic acid, 19 mg (0.14 mmol) ofN-hydroxy-benzotriazole and 100 μl (0.57 mmol) of diisopropylethylamineare dissolved in 3.5 ml of dichloromethane, mixed with a solution of 67mg (0.33 mmol) of dicyclohexylcarbodiimide in 0.5 ml of dichloromethaneand stirred for 64 hours at room temperature. For working-up, theprecipitated urea is filtered off, and the residue that remains afterremoval of the solvent i.vac. is chromatographed (23 g of silica gel 60,dichloromethane/acetone/hexane 1:1:1→acetone/hexane 2:1). 98 mg (80%) ofN-(N-(N-(N-(3-phenyl -butenoyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,21-dihydroxy-3,20-dioxo-6α-methyl-17-propionyloxy-pregna-1,4-dien-21-yl] ester, and, after recrystallization, 81 mg (66%) of crystallinecompound from ethyl acetate/hexane are obtained.

Melting point sintering starting from 154°, melt at 191° C., HPLC:97.2-97.9%, ASA: Ala 1.89 Pro 1.13 Val 0.98, racemic test (GC): D-Ala10.2% D-Pro 3.6% D-Val 2.5%.

Example 14

N-(N-(N-(N-(4-Chlorobenzenesulfonyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,21-dihydroxy-3,20-dioxo-6α-methyl-17-propionyloxy-pregna-1,4-dien-21-yl]ester (Cbs -Ala-Ala-Pro-Val-O-MPP)

100 mg (0.11 mmol) of H-Ala-Ala-Pro-Val-O-MPP x TFA (Example 3) and 300μl (1.71 mmol) of diisopropylethylamine are dissolved in 5 ml oftetrahydrofuran, mixed with 238 mg (1.13 mmol) of4-chlorobenzenesulfonic acid chloride and stirred for 23 hours at roomtemperature. The residue that remains after removal of the solventi.vac. is chromatographed. Gradient chromatography on 25 g of silica gel60 (hexane/acetone 2:1→1:2) yields 75 mg (70%) ofN-(N-(N-(N-(4-chlorobenzenesulfonyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,21-dihydroxy-3,20-dioxo-6α-methyl-17-propionyloxy-pregna-1,4-dien-21-yl]ester, and, after recrystallization from dichloromethane/diisopropylether, 47 mg (44%) of crystalline product.

Melting point sintering starting from 151°, melt at 195° C., HPLC:97.2-98.0%, ASA: Ala 1.60 Pro 1.28 Val 1.12, racemic test (GC): D-Ala2.8% D-Pro 1.2% D-Val 0.8%

Example 15

N-(N-(N-(N-(2,2-Dimethylpropionyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine [11β,21-dihydroxy-3,20-dioxo-6α-methyl-17-propionyloxy-pregna-1,4-dien-21-yl] ester (Piv-Ala-Ala-Pro-Val-O-MPP)

150 mg (0.17 mmol) of H-Ala-Ala-Pro-Val-O-MPP x TFA (Example 3) and 60μl (0.34 mmol) of diisopropylethylamine are dissolved in 6 ml ofdichloromethane, mixed with 20 μl (0.18 mmol) of pivaloyl chloride andstirred for 25 hours at room temperature. The residue that remains afterremoval of the solvent i.vac. is directly chromatographed. Gradientchromatography (24 g of silica gel 60, hexane→hexane/acetone 7:3) yields102 mg (70%) of slightly contaminatedN-(N-(N-(N-(2,2-dimethylpropionyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,21-dihydroxy-3,20-dioxo-6α-methyl-17-propionyloxy-pregna-1,4-dien-21-yl]ester. A second chromatography (Lichroprep Si60A pre-packed column,hexane/dichloromethane 1:1→hexane/dichloromethane/methanol 30:60:10)yields 92 mg, 83 mg of crystalline compound consisting ofdichloromethane/diisopropyl ether.

Sintered starting from 157° C. melting point 177° C. HPLC: 98.7%, ASA:Ala 1.90 Pro 1.10 Val 1.00, racemic test (GC): D-Ala 2.6% D-Pro 1.8%D-Val 0.6%.

Example 16

N-(N-(N-(N-(3-(Carbazol-9-yl)-propionyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,21-dihydroxy-3,20-dioxo-6α-methyl-17-propionyloxy-pregna-1,4-dien-21-yl]ester (Cbp-Ala-Ala-Pro-Val -O-MPP)

150 mg (0.17 mmol) of H-Ala-Ala-Pro-Val-O-MPP x TFA (Example 3), 61 mg(0.26 mmol) of carbazolepropionic acid, 35 mg (0.26 mmol) ofN-hydroxy-benzotriazole and 122 μl (070 mmol) of diisopropylethylamineare dissolved in 5 ml of dichloromethane, mixed with 65 mg (0.34 mmol)of ethyl-3(dimethylamino)propylcarbodiimide (EDC) and stirred for 67hours at room temperature. For working-up, the solvent is removed i.vac.and the residue that remains is chromatographed (23 g of silica gel 60,acetone/hexane 1:9→1:1). 140 mg (83%) ofN-(N-(N-(N-(3-(carbazol-9-yl)-propionyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,21-dihydroxy-3,20-dioxo-6α-methyl-17-propionyloxy-pregna-1,4-dien-21-yl] ester, and, after recrystallization,, 99 mg (59%) incrystalline form from acetone/hexane are obtained.

Melting point sintering starting from 158°, melt at 182° C., HPLC:95.8-96.3%, racemic test (GC): D-Ala 2.0% D-Pro 2.1% D-Val 1.4%.

Example 17

N-(N-(N-(N-(N-(Phthaloyl)-glycyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine [11β,21-dihydroxy-3,20-dioxo-6α-methyl-17-propionyloxy-pregna-1,4-dien-21-yl] ester (Pth-Gly-Ala-Ala-Pro-Val-O-MPP)

120 mg (0.14 mmol) of H-Ala-Ala-Pro-Val-O-MPP x TFA (Example 3), 31 mg(0.15 mmol) of phthaloylglycine, 20 mg (0.15 mmol) ofN-hydroxy-benzotriazole and 48 μl (0.27 mmol) of diisopropylethylamineare dissolved in 2 ml of dichloromethane, mixed with a solution of 56 mg(0.27 mmol) of dicyclohexylcarbodiimide in 1 ml of dichloromethane andstirred for 17 hours at room temperature. For working-up, theprecipitated urea is filtered off and the residue that remains afterremoval of the solvent i.vac. is chromatographed (gradientchromatography, 25 g of silica gel 60, hexane/acetone 1:1→1:2). 68 mg(52%) of N-(N-(N-(N-(N-(phthaloyl)-glycyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,21-dihydroxy-3,20-dioxo-6α-methyl-17-propionyloxy-pregna-1,4-dien-21-yl]ester and from this, 66 mg (51%) of crystalline product fromdichloromethane/diisopropyl ether are obtained.

Melting point (dec.) with gas generation starting from 165° C., HPLC:97.2-98.4, ASA: Ala 1.89 Pro 1.12 Val 0.99, racemic test (GC): D-Ala1.1% D-Pro 0.8% D-Val 0.8% Gly identified.

Example 18

N-(N-(N-(N-(N-((9H-Fluoren-9-ylmethoxy)carbonyl)-2-amino-2-methyl-propionyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,21-dihydroxy-3,20-dioxo-6α-methyl-17-propionyloxy-pregna-1,4-dien-21-yl] ester (Fmoc-Aib-Ala-Ala-Pro-Val-O-MPP)

200 mg (0.14 mmol) of H-Ala-Ala-Pro-Val-O-MPP x TFA (Example 3), 120 mg(0.34 mmol) of Fmoc-Aib-NCA and 200 μl (1.14 mmol) ofdiisopropylethylamine are dissolved in 10 ml of dichloromethane andstirred for 23 hours at room temperature. For working-up, the solvent isremoved i.vac. and the residue that remains is chromatographed (gradientchromatography, 25 g of silica gel 60, hexane→hexane/acetone 1:2). 135mg (55%) of N-(N-(N-(N-(N-((9H-fluoren-9-ylmethoxy)carbonyl)-2-amino-2-methyl-propionyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,21-dihydroxy-3,20-dioxo-6α-methyl-17-propionyloxy-pregna-1,4-dien-21-yl]ester, and, after recrystallization, 102 mg (44%) after crystallizationfrom dichloromethane/diisopropyl ether that is repeated twice areobtained.

[α]_(D) =-23° (c=0.26% in chloroform), HPLC: 97-98%. Racemic test (GC):D-Ala 1.4% D-Pro 1.4% D-Val 1.7% Aib identified.

Example 19

N-(1,1-Dimethylethoxycarbonyl)-valine[11β,17,20-trihydroxy-3,20-dioxo-6α-methyl-pregna-1,4-dien-21-yl] ester(Boc-Val-O-MP)

Analogously to Example 1, 1.94 g of 6e-methylprednisolone, 1.37 g ofN-(1,1-dimethylethoxycarbonyl)-valine, 74 mg of 4-dimethylaminopyridineand 1.34 g of dicyclohexylcarbodiimide in 30 ml ofdichloromethane/dioxane 1:1 are reacted. Chromatography on silica gel(dichloromethane→dichloromethane/acetone 3:1) yields 2.58 g (87%) ofN-(1,1-dimethylethoxycarbonyl)-valine[11β,17,20-trihydroxy-3,20-dioxo-6α-methyl-pregna-1,4-dien-21-yl] ester.Crystallization from dichloromethane/diisopropyl ether.

Melting point 144°-148° C. [α]_(D) =+70° (chloroform).

Example 20

Valine [11β,17,20-trihydroxy-3,20-dioxo-6α-methyl-pregna-1,4-dien-21-yl]ester trifluoroacetate (H-Val-O-MP TFA)

806 mg of N-(1,1-dimethylethoxycarbonyl)-valine[11β,17,20-trihydroxy-3,20-dioxo-6α-methyl-pregna-1,4-dien-21-yl] ester(Example 19) is reacted under the conditions described in Example 2. 616mg (75%) of valine[11β,17,20-trihydroxy-3,20-dioxo-6α-methyl-pregna-1,4-dien-21-yl] estertrifluoroacetate is obtained.

Melting point 167°-171° C. (decomposition). [α]_(D) =+95° (methanol).

Example 21

N-(N-(N-(N-(1,1-Dimethylethoxycarbonyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine [11β,17,21-trihydroxy-3,20-dioxo-6α-methyl-pregna-1,4-dien-21-yl] ester (Boc-Ala-Ala-Pro-Val-O-MP)

Under the conditions indicated in Example 3, 1.44 g (2.46 mmol) ofvaline [11β, 17,20-trihydroxy-3,20-dioxo-6α-methyl-pregna-1,4-dien-21-yl] ester trifluoroacetate, 800 mg (2.24 mmol) ofN-(1,1-dimethylethoxycarbonyl)-alanyl-alanyl-proline, 300 mg (2.24 mmol)of hydroxybenzotriazole, 555 mg (2.69 mmol) of dicyclohexylcarbodiimideand 0.49 ml (4.48 mmol) of N-methylmorpholine are reacted.Chromatography on silica gel (dichloromethane→dichloromethane/methanol95:5) provides 1.54 g (77%) ofN-(N-(N-(N-(1,1-dimethylethoxycarbonyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,17,21-trihydroxy-3,20-dioxo-6α-methyl-pregna-1,4-dien-21-yl] ester.Crystallization from dichloromethane/diisopropyl ether.

Melting point 155°-175° C., decomposition with gas generation, [α]_(D)=-2° (c=0.5% in chloroform), HPLC: 96.5-97.5%. Racemic test (GC): D-Ala3.2% D-Pro 2.3% D-Val 1.9%. Cld: C 63.53 H 7.93 N 6.89 Fnd: C 63.52 H7.70 N 6.97

Example 22

N-(N-(N-(N-((9H-Fluoren-9-yl-methoxycarbonyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,17,21-trihydroxy-3,20-(dioxo-6α-methyl-pregna-1,4-dien-21-yl] ester(Fmoc-Ala-Ala-Pro-Val-O-MP)

510 mg (0.87 mmol) of valine[11β,17,20-trihydroxy-3,20-dioxo-6α-methyl-pregna-1,4-dien-21-yl] estertrifluoroacetate, 347 mg (0.72 mmol) of Fmoc-Ala-Ala-Pro-OH and 97 mg(0.72 mmol) of N-hydroxybenzotriazole are added to this sequence in 25ml of dichloromethane, dissolved with 160 μl (1.45 mmol) ofN-methylmorpholine. Then, a solution of 298 mg (1.45 mmol) ofdicyclohexylcarbodiimide in 5 ml of dichloromethane is mixed and stirredfor 5 hours at room temperature After cooling to -20° C. it is suctionedoff from precipitated urea, washed with diethyl ether, the combinedfiltrates are extracted with 2×50 ml of 0.5N HCl, 0.5N NaOH each, washedwith saturated sodium chloride solution and dried on sodium sulfate. Theresidue that remains after removal of the solvent i.vac. is directlychromatographed (110 g of silica gel,dichloromethane→dichloromethane/methanol 9:1). 478 mg (71%) ofN-(N-(N-(N-((9H-fluoren-9-yl-methoxycarbonyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,17,21-trihydroxy-3,20-dioxo-6α-methyl-pregna-1,4-dien-21-yl] esteris obtained. Crystallization from dichloromethane/diisopropyl etheryields 449 mg.

Melting point 169°-171° C., [α]_(D) =-2° (c=0.5% in chloroform), HPLC:96.1-96.8%. Racemic test (GC): D-Ala 2.5% D-Pro>1% D-Val 1.4%. Cld: C68.07 H 7.11 N 5.99 O 18.82 Fnd: C 67.95 H 7.54 N 5.68 O 18.74

Example 23

N-(N-(N-(L-Alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,17,21-trihydroxy-3,20-dioxo-6α-methyl-pregna-1,4-dien-21-yl] esterhydrochloride (H-Ala-Ala-Pro-Val-O-MP x HCl)

939 mg (1.15 mmol) of Boc-Ala-Ala-Pro-Val-O-MP (Example 21) is dissolvedin 10 ml of dioxane and 10 ml of HCl (4N in dioxane) is added. After 18hours at room temperature, the solvent is removed i.vac., the residue ispulverized with dichloromethane, suctioned off and the obtained crystalsare recrystallized from methanol/diisopropyl ether. 835 mg (94%) ofN-(N-(N-(L-alanyl) -L-alanyl)-L-prolyl)-L-valine[11β,17,21-trihydroxy-3,20-dioxo-6α-methyl-pregna-1,4-dien-21-yl] esterhydrochloride is obtained.

Melting point starting from 200° C. (dec), [α]_(D) =-10° (c=0.5% inmethanol), HPLC: 97%, racemic test (GC): D-Ala 2.7% D-Pro 1.7% D-Val>1%.

Example 24

N-(N-(N-(N-(Phenylcarbonyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,17,21-trihydroxy-3,20-dioxo-6α-methyl-pregna-1,4-dien-21-yl] ester(Bz-Ala-Ala-Pro-Val-O-MP)

370 mg (0.46 mmol) of Boc-Ala-Ala-Pro-Val-O-MP (Example 21) is dissolvedin 3 ml of dioxane and 4 ml of HCl (4N in dioxane) is added. After 16hours at room temperature, the solvent is removed i.vac., dissolved in 4ml of dichloromethane, mixed with 175 μl (1.00 mmol) ofdiisopropylethylamine and 58 μl (0.50 mmol) of benzoyl chloride andstirred for 17 hours at room temperature. For working-up, the solvent isremoved i.vac. and the residue that remains is crystallized fromdichloromethane/diisopropyl ether. 139 mg(37% ) ofN-(N-(N-(N-(phenylcarbonyl)-L-alanyl)-L -alanyl)-L-prolyl)-L-valine[11β,17,21-trihydroxy-3,20-dioxo-6α-methyl-pregna-1,4-dien-21-yl] esteris obtained.

HPLC: 97.1-97.7%, racemic test (GC): D-Ala 3.8% D-Pro 2.7% D-Val 2.6%.

Example 25

N-(N-(N-(N-((Phenylmethoxy)carbonyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine [11β,17,21-trihydroxy-3,20-dioxo-6α-methyl-pregna-1,4-dien-21-yl] ester (Z-Ala-Ala-Pro-Val-O-MP)

200 mg (0.27 mmol) of H-Ala-Ala-Pro-Val-O-MP x HCl (Example 23) and 37μl (0.27 mmol) of triethylamine are dissolved in 10 ml ofdichloromethane, mixed with 42 μl (0.30 mmol) of benzyl chloroformateand stirred for 36 hours at room temperature. For working-up, it isdiluted with 20 ml of dichloromethane and washed with 2N potassiumhydrogen sulfate solution and saturated sodium chloride solution, driedon sodium sulfate and concentrated by evaporation i.vac. The residuethat remains after removal of the solvent i.vac. is chromatographed.Gradient chromatography on 110 g of silica gel 60(dichloromethane→dichloromethane/methanol 9:1) yields 156 mg (69%) ofN-(N-(N-(N-((phenylmethoxy)-carbonyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,17,21-trihydroxy-3,20-dioxo-6α-methyl-pregna-1,4-dien-21-yl] esterand, after recrystallization, 123 mg (54%) in crystalline form frommethanol/diisopropyl ether.

HPLC: 98.7-99.7%, ASA: Ala 2.00 Pro 1.01 Val 0.99, racemic test (GC):D-Ala 2.2% D-Pro <1% D-Val 1.1%.

Example 26

N-(N-(N-(N-(Valeroyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,17,21-trihydroxy-3,20-dioxo-6α-methyl-pregna-1,4-dien-21-yl] ester(Valeroyl-Ala-Ala-Pro-Val-O-MP)

200 mg (0.27 mmol) of H-Ala-Ala-Pro-Val-O-MP x HCl 41c 33 μl (0.30 mmol)of valeric acid, 41 mg (0.30 mmol) of N-hydroxybenzotriazole and 66 μl(0.60 mmol) of N-methylmorpholine are dissolved in 20 ml ofdichloromethane, mixed with a solution of 123 mg (0.60 mmol) ofdicyclohexylcarbodiimide in 5 ml of dichloromethane and stirred for 105hours at room temperature. For working-up, the precipitated urea isfiltered off and the filtrate is concentrated by evaporation i.vac.Gradient chromatography of the amount of raw material of 110 g of silicagel 60 (hexane→hexane/acetone 6:4) yields 139 mg (65%) of N-(N-(N-(N-(valeroyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,17,21-trihydroxy-3,20-dioxo-6α-methyl-pregna-1,4-dien-21-yl] ester,74 mg (35%) in crystalline form from methanol/diisopropyl ether.

HPLC: 96.3-97.0%, racemic test (GC): D-Ala 2.5% D-Pro 2.4% D-Val<1%.

ZK 162494 AZ 204436 ON 128

Example 27

(2S)-2-((1,1-Dimethylethoxycarbonyl)-amino)-3-methyl-butyric acid[11β,17,21-trihydroxy-3,20-dioxo-pregn-4-en-21-yl] ester (Boc -Val-O-HC)

A solution of 3.63 g (10 mmol) of hydrocortisone in 150 ml ofdichloromethane is mixed with 2.34 g (10.8 mmol) of N-(tert-butoxycarbonyl)-valine, 500 mg (4.1 mmol) of 4-dimethylaminopyridineand 3.1 g (15 mmol) of dicyclohexylcarbodiimide. The solution is stirredfor 3 hours at room temperature, the precipitate that is produced issuctioned off and washed with dichloromethane. The filtrate isconcentrated by evaporation in a vacuum. The chromatography on silicagel (hexane→hexane/ethyl acetate 1:1) provides 2.87 g (51%) of(2S)-2-((1,1-dimethylethoxycarbonyl)-amino)-3-methylbutyric acid[11β,17,21-trihydroxy-3,20-dioxo-pregn-4-en-21-yl] ester.Crystallization from dichloromethane/diisopropyl ether.

Melting point 169° C., [α]_(D) =+97° (chloroform).

Example 28

(2S)-2-Amino-3-methyl-butyric acid[11β,17,21-trihydroxy-3,20-dioxo-pregn-4-en-21-yl] estertrifluoroacetate (H-Val-O-HC-TFA)

500 mg (0.89 mmol) of (2S)-2-((1,1-dimethylethoxycarbonyl)-amino)-3-methyl-butyric acid [11β,17,21-trihydroxy-3,20-dioxo-pregn-4-en-21-yl] ester (Example 27) is mixed with 1 ml oftrifluoroacetic acid and stirred for 10 minutes at room temperature.Then, the trifluoroacetic acid is evaporated in a vacuum. The residue ismixed with stirring with a little diethyl ether, the white precipitatethat is produced is suctioned off and dried. 385 mg (76%) of(2S)-2-amino-3-methyl-butyric acid[11β,17,21-trihydroxy-3,20-dioxo-pregn-4-en-21-yl] estertrifluoroacetate is obtained.

Melting point 188° C.

Example 29

N-(N-(N-(N-(9H-Fluoren-9-yl-methoxycarbonyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine [11β,17,21-trihydroxy-3,20-dioxo-pregn-4-en -21-yl]ester (Fmoc-Ala-Ala-Pro-Val-O-HC)

A solution of 350 mg (0.62 mmol) of (2S)-2-amino-3-methylbutyric acid[11β,17,21-trihydroxy-3,20-dioxo-pregn-4-en-21-yl] estertrifluoroacetate (Example 28) in 30 ml of dichloromethane is mixed with350 mg (0.73 mmol) ofN-(9-fluorenylmethoxycarbonyl)-alanyl-alanyl-proline, 100 mg (0.74 mmol)of hydroxybenzotriazole, 150 mg (0.72 mmol) of dicyclohexylcarbodiimideand 0.07 ml (0.63 mmol) of N-methylmorpholine, and it is stirred for 2hours at room temperature. For working-up, it is filtered off fromdicyclohexylurea, rewashed with diethyl ether. The combined organicphases are washed with 50 ml of 0.5N HCl, 0.5N NaOH and saturated sodiumchloride solution in each case and dried on sodium sulfate. Evaporationof the solvent in a vacuum yields the crude product. The chromatographyon silica gel (hexane→hexane/acetone 1:1) yields 405 mg (72%) ofN-(N-(N-(N-(9H-fluoren-9-yl-methoxycarbonyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,17,21-trihydroxy-3,20-dioxo-pregn-4-en-21-yl] ester.

Crystallization from hexane/ethyl acetate.

Melting point 193° C.

Example 30

(2S)-2-((1,1-Dimethylethoxycarbonyl)-amino)-3-methyl-butyric acid[11β,21-dihydroxy-6α-fluoro-16α-methyl-3,20-dioxo-pregna-1,4-dien-21-yl]ester (Boc-Val-O-FC)

26 mmol of fluocortolone (FC) is reacted with 32 mmol of Boc-valine(Boc-Val-OH), 2.9 mmol of 4-dimethylaminopyridine and 34 mmol ofdicyclohexylcarbodiimide in dichloromethane/dioxane 3:2. Chromatography(ethyl acetate/hexane 2:1): F₁ 9.3 g of pure product, F₂ 4.8 g ofslightly contaminated product. (Total yield 92%) crystallization of F₁from ethyl acetate/hexane yields 7.0 g (46%) of(2S)-2-((1,1-dimethylethoxycarbonyl)-amino)-3-methyl-butyric acid[11β,21-dihydroxy-6α-fluoro-16α-methyl-3,20-dioxo-pregna-1,4-dien-21-yl]ester.

Melting point 166°-168° C., [α]_(D) =+85° (c=0.55 in chloroform) . Cld:C 66.88 H 7.89 N 2.44 F 3.31 Fnd: C 66.90 H 7.82 N 2.73 F 3.29

Example 31

(2S)-2-Amino-3-methyl-butyric acid [11β,21-dihydroxy-6α-fluoro-16α-methyl-3,20-dioxo-pregna-1,4-dien-21-yl] ester trifluoroacetate(H-Val-O-FC x TFA)

6.4 g (11 mmol) of Boc-Val-O-FC (Example 29) is reacted withtrifluoroacetic acid/dichloromethane analogously to Example 2.Crystallization from dichloromethane/diisopropyl ether (ultrasonic bath)yields 5.6 g (86%) of (2S)-2-amino-3-methylbutyric acid[11β,21-dihydroxy-6α-fluoro-16α-methyl-3,20-dioxo-pregna-1,4-dien-21-yl] ester trifluoroacetate.

Melting point 135°-138° C.

Example 32

N-(N-(N-(N-((1,1-Dimethyl)-ethoxycarbonyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[6α-fluoro-11β,21-dihydroxy-16α-methyl-3,20dioxo-pregna-1,4-dien-21-yl]ester (Boc-Ala-Ala-Pro-Val-O-FC)

884 mg (1.50 mmol) of H-Val-O-FC x TFA (Example 31), 500 mg (1.40 mmol)of Boc-Ala-Ala-Pro-OH and 168 mg (1.40 mmol) of N-hydroxybenzotriazoleare dissolved in this sequence in 50 ml of dichloromethane and 330 μl(3.00 mmol) of N-methylmorpholine is added. Then, a solution of 289 mg(1.40 mmol) of dicyclohexylcarbodiimide in 2 ml of dichloromethane isadded and stirred for 2 hours at room temperature. Then, it is suctionedoff from precipitated urea, the filtrate is concentrated by evaporationand filtered again. Chromatography of this amount of raw material (300 gof silica gel, hexane/acetone 1:1) yields 861 mg (76%) ofN-(N-(N-(N-((1,1-dimethyl) ethoxycarbonyl) -L-alanyl)-L-alanyl)-L-prolyl)-L-valine[6α-fluoro-11β,21-dihydroxy-16α-methyl-3,20-dioxo-pregna-1,4-dien-21-yl]ester. 737 mg of pure product is obtained [one or more words missing]recrystallization from dichloromethane/diisopropyl ether.

Melting point starting from 136° C. decomposition [α]_(D) =0° (c=0.5% inchloroform) , HPLC: 98%. Racemic test (GC): D-Ala 3.0% D-Pro 1.9%D-Val<1%.

Example 33

N-(N-(N-(N-(9H-Fluoren-9-yl-methoxycarbonyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[6α-fluoro-11β,21-dihydroxy-16α-methyl-3,20-dioxo-pregna-1,4-dien-21-yl]-pregna-1,4-dien-21-yl]ester (Fmoc -Ala-Ala-Pro-Val-O-FC)

442 mg (0.75 mmol) of H-Val-O-TCA x TFA (Example 31), 336 mg (0.70 mmol)of Fmoc-Ala-Ala-Pro-OH and 84 mg (0.70 mmol) of N-hydroxybenzotriazoleare dissolved in this sequence in 20 ml of dichloromethane, and 155 μl(1.50 mmol) of N-methylmorpholine is added. Then, a solution of 145 mg(0.70 mmol) of dicyclohexylcarbodiimide in 2 ml of dichloromethane isadded and stirred for 6 hours at room temperature. Then, it is suctionedoff from precipitated urea, the filtrate is concentrated by evaporationand filtered again. Chromatography of this amount of raw material (300 gof silica gel, dichloromethane→dichloromethane/acetone 1:1) yields 298mg (=44%) of N-(N-(N-(N-(9H-fluoren-9-yl-methoxycarbonyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine [6α-fluoro-11β,21-dihydroxy-16α-methyl-3,20-dioxo-pregna-1,4-dien-21-yl]-pregna-1,4-dien-21-yl] ester. Recrystallizationfrom dichloromethane/diisopropyl ether yields 158 mg of pure substance.This material also contained several contaminants and is furtherpurified by HPLC (Novapak, MeCN/10 mmol of ammonium hydrogen carbonate60:40).

Melting point starting from 158° C. decomposition, [α]_(D) =+1° (c=0.5%in chloroform), HPLC: 99%. Racemic test (GC): D-Ala 1.0% D-Pro 1.4%D-Val 3.5%.

Example 34

N-(N-(N-(L-Alanyl)-L-alanyl)-L-prolyl)-L-valine [6α-fluoro-11β,21-dihydroxy-16α-methyl-3,20-dioxo-pregna-1,4-dien-21-yl]-pregna-1,4-dien-21-yl]ester hydrochloride (H-Ala-Ala-Pro-Val-O-FC x HCl)

113 mg (0.14 mmol) of Boc-Ala-Ala-Pro-Val-O-FC (Example 32) is dissolvedin 1 ml of hydrochloric acid dioxane (4N HCl in dioxane). After 6 hoursat room temperature, the solvent is removed i.vac., the residue isrecrystallized from dichloromethane/diisopropyl ether. 117 mg (>100%) ofN-(N-(N -(L-alanyl)-L-alanyl)-L-prolyl)-L-valine[6α-fluoro-11β,21-dihydroxy-16α-methyl-3,20-dioxo-pregna-1,4-dien-21-yl]-pregna-1,4-dien-21-yl] ester hydrochloride is obtained.

HPLC: 98.3-99.3%, racemic test (GC): D-Ala 6.4% D-Pro 3.4% D-Val 1.5%.

Example 35

N-(N-(N-(N-(Phenylcarbonyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[6α-fluoro-11β,21-dihydroxy-16α-methyl-3,20-dioxo-pregna-1,4-dien-21-yl]-pregna-1,4-dien-21-yl] ester (Bz-Ala-Ala-Pro -Val-O-FC)

105 mg (0.13 mmol) of H-Ala-Ala-Pro-Val-O-FC x HCl (Example 34) isdissolved in 3 ml of dichloromethane and 3 ml of triethylamine, mixedwith 23 μl (0.20 mmol) of benzoyl chloride and stirred for 8 hours atroom temperature. For working-up, it is washed with 1N HCl and water,dried on sodium sulfate and concentrated by evaporation i.vac. Theresidue that remains after removal of the solvent i.vac. is directlychromatographed. Gradient chromatography on 50 g of silica gel 60(dichloromethane →dichloromethane/methanol 95:5) yields 96 mg (84%) ofN-(N-(N -(N-(phenylcarbonyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[6α-fluoro-11β,21-dihydroxy-16α-methyl-3,20-dioxo-pregna-1,4-dien-21-yl]-pregna-1,4-dien-21-yl]ester, 75 mg (66%) yields 36 mg of pure compound after recrystallizationfrom dichloromethane/diisopropyl ether.

HPLC: 96.1-96.9%, racemic test (GC): D-Ala 8.1% D-Pro<1% D-Val 1.4%.

Example 36

(2S)-2-((1,1-Dimethylethoxycarbonyl)-amino)-3-methyl-butyric acid[11β,21-dihydroxy-3,20-dioxo-9-fluoro-16β-methyl-17-valeroyloxy-pregna-1,4-dien-21-yl] ester (Boc-Val-O-BMV)

21 mmol of betamethasone-17-valerate is reacted with 25 mmol ofBoc-valine (Boc-Val-OH), 2.5 mmol of 4-dimethylaminopyridine and 27 mmolof dicyclohexylcarbodiimidazole in dichloromethane/dioxane 1:1analogously to Example 1. Chromatography (1. ethyl acetate/hexane 2:1and 2. ethyl acetate/hexane 1:1) yields 10.1 g (73%) of pure(2S)-2-((1,1-dimethylethoxycarbonyl)-amino)-3-methyl-butyric acid[11β,21-dihydroxy-3,20-dioxo-9-fluoro-16β-methyl-17-valeroyloxy-pregna-1,4-dien-21-yl] ester as foam.

[α]_(D) =+51° (c=0.5% in chloroform). Cld: C 65.86 H 7.92 N 2.08 F 2.82Fnd: C 65.53 H 7.72 N 2.24 F 2.69

Example 37

(2S)-2-Amino-3-methyl-butyric acid [11β,21-dihydroxy-3,20-dioxo-9-fluoro-16β-methyl-17-valeroyloxy-pregna-1,4-dien-21-yl] estertrifluoroacetate (H-Val-O-BMV x TFA)

8.5 g (13 mmol) of Boc-Val-O-BMV (Example 36) is reacted withtrifluoroacetic acid/dichloromethane analogously to Example 2. Thecompound is not crystalline and is further used as crude product. C-NMRcorresponds to the expected structure.

Example 38

N-(N-(N-(N-((1,1-Dimethyl)ethoxycarbonyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[9α-fluoro-11β,21-dihydroxy-16β-methyl-3,20-dioxo-17-valeroyloxy-pregna-1,4-dien-21-yl]ester (Boc-Ala-Ala -Pro-Val-O-BMV)

1.04 g (1.50 mmol) of H-Val-O-BMV x TFA (Example 37), 500 mg (1.40 mmol)of Boc-Ala-Ala-Pro-OH and 168 mg (1.40 mmol) of Nhydroxybenzotriazoleare dissolved in this sequence in 50 ml of dichloromethane, and 330 μl(3.00 mmol) of N-methylmorpholine is added. Then, a solution of 289 mg(1.40 mmol) of dicyclohexylcarbodiimide in 2 ml of dichloromethane isadded and stirred for 20 hours at room temperature. Then, it issuctioned off from precipitated urea, the filtrate is concentrated byevaporation and filtered again. Chromatography of this amount of rawmaterial (300 g of silica gel, hexane/acetone 1:1) yields 806 mg(63% )of N-(N-(N-(N-((1,1-dimethyl)ethoxycarbonyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[9α-fluoro-11β,21-dihydroxy-16β-methyl-3,20-dioxo-17-valeroyloxy-pregna-1,4-dien-21-yl] ester. Crystallization from dichloromethane/diisopropyl ether 542 mg(42%).

Melting point starting from 137° C. decomposition , [α]_(D) =0° C.(c=0.5% in chloroform), HPLC: 98.1-99.2%. Racemic test (GC): D-Ala 2.1%D-Pro 2.2% D-Val 0.6%.

Example 39

N-(N-(N-(N-(9H-Fluoren-9-yl-methoxycarbonyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[9α-fluoro-11β,21-dihydroxy-16β-methyl-3,20-dioxo-17-valeroyloxy-pregna-1,4-dien-21-yl]ester (Fmoc-Ala-Ala -Pro-Val-O-BMV)

689 mg (1.00 mmol) of H-Val-O-BMV x TFA (Example 37), 384 mg (0.80 mmol)of Fmoc-Ala-Ala-Pro-OH and 96 mg (0.80 mmol) of N-hydroxybenzotriazoleare dissolved in this sequence in 20 ml of dichloromethane, and 155 μl(1.50 mmol) of N-methylmorpholine is added. Then, a solution of 145 mg(0.70 mmol) of dicyclohexylcarbodiimide in 2 ml of dichloromethane isadded and stirred for 6 hours at room temperature. Then, it is suctionedoff from precipitated urea, the filtrate is concentrated by evaporationand filtered again. The obtained solution is diluted withdichloromethane, extracted with 0.5N NaOH and 0.5N HCl, dried on sodiumsulfate and concentrated by evaporation i.vac. Chromatography of theamount of raw material (300 g of silica gel, dichloromethane/acetone1:1) yields 232 mg (28%) of N-(N-(N-(N-(9H-fluoren-9-yl-methoxy-carbonyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[9α-fluoro-11β,21-dihydroxy-16β-methyl-3,20-dioxo-17-valeroyloxy-pregna-1,4-dien-21-yl]ester, and, after crystallization from dichloromethane/diisopropylether, 200 mg of pure product.

Melting point starting from 143° C. decomposition, [α]_(D) =-13° (c=0.5%in chloroform), HPLC: 95.4-99%. ASA: Ala 1.98 Pro 0.99 Val 1.03, racemictest (GC): D-Ala 8.0% D-Pro 4.7% D-Val 2.6%.

Example 40

N-(N-(N-(L-Alanyl)-L-alanyl)-L-prolyl)-L-valine [9α-fluoro-11β,21-dihydroxy-16β-methyl-3,20-dioxo-17-valeroyloxy-pregna-1,4-dien-21-yl]ester hydrochloride (H-Ala-Ala-Pro-Val-O-BMV x HCl)

137 mg (0.15 mmol) of Boc-Ala-Ala-Pro-Val-O-BMV (Example 38) isdissolved in 1 ml of hydrochloric acid dioxane (4N HCl in dioxane).After 6 hours at room temperature, the solvent is removed i.vac. and theresidue is recrystallized from dichloromethane/diisopropyl ether. 114 mg(89%) of N-(N-(N-(L -alanyl)-L-alanyl)-L-prolyl)-L-valine[9α-fluoro-11β,21-dihydroxy16β-methyl-3,20-dioxo-17-valeroyloxy-pregna-1,4-dien-21-yl] esterhydrochloride is obtained.

HPLC: 97.0-99.2%, racemic test (GC): D-Ala 4.9% D-Pro 3.8% D-Val 1.1%.

Example 41

N-(N-(N-(N-(Phenylcarbonyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[9α-fluoro-11β,21-dihydroxy-16β-methyl-3,20-dioxo-17-valeroyloxy-pregna-1,4-dien-21-yl]ester (Bz-Ala-Ala-Pro-Val-O-BMV)

102 mg (0.12 mmol) of H-Ala-Ala-Pro-Val-O-BMV x HCl 40 c is dissolved in3 ml of dichloromethane, mixed with 21 μl (0.18 mmol) of benzoylchloride and 356 μl (2.50 mmol) of diisopropylethylamine and stirred for5 hours at room temperature. For working-up, it is washed with 1N HCland water, dried on sodium sulfate and concentrated by evaporationi.vac. The residue that remains after removal of the solvent i.vac. isdirectly chromatographed. Gradient chromatography on 50 g of silica gel60 (dichloromethane→dichloromethane/methanol 95:5) yields 79 mg (72%) ofN-(N-(N-(N-(phenylcarbonyl)-L -alanyl)-L-alanyl)-L-prolyl)-L-valine[9α-fluoro-11β,21-dihydroxy-16β-methyl-3,20-dioxo-17-valeroyloxy-pregna-1,4-dien-21-yl] ester and,after recrystallization, 56 mg (51%) of crystalline product from ethylacetate/hexane.

HPLC: 95% racemic test (GC): D-Ala 12.1% D-Pro 3.1% D-Val 1.4%.

Example 42

(2S)-2-((1,1-Dimethylethoxycarbonyl)-amino)-3-methyl-butyric acid[11β,21-dihydroxy-3,20-dioxo-9-fluoro-16α,17-[(1-methylethylidene)bis(oxy)]-pregna-1,4-dien-21-yl]ester (Boc -Val-O-TCA)

11 mmol of triamcinolone-acetonide (TCA) is reacted with 13 mmol ofBoc-valine (Boc-Val-OH), 1.2 mmol of DMAP and 14 mmol of DCC indichloromethane/dioxane 1:1 analogously to Example 1. Chromatography(ethyl acetate/hexane 2:1) yields 3.72 g (53%) of(2S)-2-((1,1-dimethylethoxycarbonyl)-amino)-3-methyl-butyric acid[11β,21-dihydroxy-3,20-dioxo-9-fluoro-16α,17-[(1-methylethylidene)bis(oxy)]-pregna-1,4-dien-21-yl]ester. Repeated crystallization from ethyl acetate/hexane provides 5.52g (52%) of pure product.

Melting point 238-240° C., [α]_(D) =+75° (c=0.5% in chloroform) . Cld: C64.54 H 7.49 N 2.21 F 3.00 Fnd: C 64.23 H 7.21 N 2.35 F 2.88

Example 43

(2S)-2-Amino-3-methyl-butyric acid [11β,21-dihydroxy-3,20-dioxo-9-fluoro-16α,17-[(1-methylethylidene)bis(oxy)]-pregna-1,4-dien -21-yl]ester trifluoroacetate (H-Val-O-TCA x TFA)

2.5 g (4.0 mmol) of Boc-Val-O-TCA (Example 42) is reacted withtrifluoroacetic acid/dichloromethane analogously to Example 2.Crystallization from dichloromethane/diisopropyl ether yields 2.58 g(100%) of (2S)-2-amino-3-methyl-butyric acid[11β,21-dihydroxy-3,20-dioxo-9-fluoro-16α, 17- [(1-methylethylidene)bis(oxy)]-pregna-1,4-dien-21-yl] ester trifluoroacetate.

Example 44

N-(N-(N-(N-((1,1-Dimethyl)ethoxycarbonyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,21-dihydroxy-3,20-dioxo-9-fluoro-16α,17-[(1-methylethylidene) his(oxy)]-pregna-1,4-dien-21-yl] ester (Boc-Ala-Ala-Pro-Val-O-TCA)

900 mg (1.50 mmol) of H-Val-O-TCA x TFA (Example 43), 500 mg (1.40 mmol)of Boc-Ala-Ala-Pro-OH and 168 mg (1.40 mmol) of N-hydroxybenzotriazoleare dissolved in this sequence in 50 ml of dichloromethane, and 300 μl(3.00 mmol) of N-methylmorpholine is added. Then, a solution of 289 mg(1.40 mmol) of dicyclohexylcarbodiimide in 2 ml of dichloromethane isadded and stirred for 2 hours at room temperature. Then, it is suctionedoff from precipitated urea, the filtrate is concentrated by evaporationand filtered again. Chromatography of this amount of raw material (300 gof silica gel, hexane/acetone 1:1) yields 935 mg (71%) ofN-(N-(N-(N-((1,1-dimethyl)ethoxycarbonyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,21-dihydroxy-3,20-dioxo-9-fluoro-16α,17-[(1-methylethylidene)bis(oxy)]-pregna-1,4-dien-21-yl]ester. Repeated crystallization from dichloromethane/diisopropyl etherprovides 817 mg of pure product.

Melting point starting from 151° C. decomposition [α]_(D) =-1° (c=0.26%in chloroform), HPLC: 93-95%, racemic test (GC): D-Ala 3.0% D-Pro 1.9%D-Val<1%.

Example 45

N-(N-(N-(N-(9H-Fluoren-9-yl-methoxycarbonyl)-L-alanyl)-L-alanyl)L-prolyl)-L-valine[11β,21-dihydroxy-3,20-dioxo-9-fluoro-16α,17-(1-methylethylidene)bis(oxy)]-pregna-1,4-dien-21-yl]ester (Fmoc-Ala-Ala-Pro-Val-O-TCA)

485 mg (0.75 mmol) of H-Val-O-TCA x TFA (Example 43), 336 mg (0.70 mmol)of Fmoc-Ala-Ala-Pro-OH and 84 mg (0.70 mmol) of N-hydroxybenzotriazoleare dissolved in this sequence in 20 ml of dichloromethane and 155 μl(1.50 mmol) of N-methylmorpholine is added. Then, a solution of 145 mg(0.70 mmol) of dicyclohexylcarbodiimide in 2 ml of dichloromethane isadded and stirred for 12 hours at room temperature. Then, it issuctioned off from precipitated urea, the filtrate is concentrated byevaporation and filtered again. Chromatography of this amount of rawmaterial (300 g of silica gel, dichloromethane→dichloromethane/acetone1:1) yields 215 mg (30%) of N-(N-(N-(N-(9H-fluoren-9-yl-methoxycarbonyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,21-dihydroxy-3,20-dioxo-9-fluoro-16α,17-[(1methylethylidene)bis(oxy)]-pregna-1,4-dien-21-yl]ester, which does not yield any pure product after crystallization fromdichloromethane/diisopropyl ether and ethyl acetate/hexane. Also, asecond chromatography (100 g of silica gel, hexane/acetone 1:1) is alsounsuccessful. The remaining 50 mg is purified by preparative HPLC(Novapak, MeCN/10 mmol of ammonium hydrogen carbonate 60:40).

HPLC: 99.3-99.6%. Racemic test (GC): D-Ala 1.0% D-Pro 0.8% D-Val 3.2%.

Example 46

N-(N-(N-(L-Alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,21-dihydroxy-3,20-dioxo-9-fluoro-16α,17-[(1-methylethylidene)bis(oxy)]-pregna-1,4-dien-21-yl]ester hydrochloride (H-Ala-Ala-Pro-Val-O-TCA x HCl)

131 mg (0.15 mmol) of Boc-Ala-Ala-Pro-Val-O-TCA (Example 44) isdissolved in 1 ml of hydrochloric acid dioxane (4N HCl in dioxane).After 6 hours at room temperature, the solvent is removed i.vac. and theresidue is recrystallized from dichloromethane/diisopropyl ether. 115 mg(95%) of N-(N-(N-(L -alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,21-dihydroxy-3,20-dioxo-9-fluoro-16α,17(1-methyl-ethylidene)bis(oxy)]-pregna-1,4-dien-21-yl]ester hydrochloride is obtained.

Racemic test (GC): D-Ala 2.1% D-Pro<1% D-Val 0.8%.

Example 47

N-(N-(N-(N-(Phenylcarbonyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,21-dihydroxy-3,20-dioxo-9-fluoro-16α,17-[(1-methylethylidene)bis(oxy)]-pregna-1,4-dien-21-yl]ester (Bz-Ala -Ala-Pro-Val-O-TCA)

105 mg (0.13 mmol) of H-Ala-Ala-Pro-Val-O-TCA x HCl (Example 46) isdissolved in 3 ml of dichloromethane and 3 ml of triethylamine, mixedwith 23 μl (0.20 mmol) of benzoyl chloride and stirred for 8 hours atroom temperature. [or working-up, it is washed with 1N HCl and water,dried on sodium sulfate and concentrated by evaporation i.vac. Theresidue that remains after removal of the solvent i.vac. is directlychromatographed. Gradient chromatography on 50 g of silica gel 60(dichloromethane →dichloromethane/methanol 95:5) yields 85 mg (76%) ofN-(N-(N -(N-(phenylcarbonyl)-L-alanyl)-L-alanyl)-L-prolyl)-L-valine[11β,21-dihydroxy-3,20-dioxo-9-fluoro-16β,17-(1-methylethylidene)bis(oxy)]-pregna-1,4-dien-21-yl]ester and, after recrystallization, 56 mg (49%) of crystalline pureproduct from ethyl acetate/hexane.

Biological Data

1. Binding to the Glucocorticoid Receptor

The binding of the substances according to the invention to the ratglucocorticoid receptor is determined by competitive displacement oftritiated dexamethasone from the receptor binding. Differentconcentrations of the glucocorticoid esters are incubated for 2 hours at4° C. in the presence of a constant concentration of tritium-labeleddexamethasone with rat thymus cytosol. After the incubation time, thenon-protein-bound steroids are adsorbed on activated carbon and theactivated carbon is centrifuged off. The radioactivity in thesupernatant is used as a measurement for the amount of receptor-bounddexamethasone.

From the measurements, the concentrations of the respective corticoidderivative are calculated, which are necessary to displace 50% of thelabeled dexamethasone from the receptor binding. Competition factor (KF)is the quotient of these calculated concentrations for the testsubstance and that of dexamethasone. The better a compound binds to thereceptor, the lower its KF is.

As can be seen from Table 1, the glucocorticoid-oligopeptide-estersaccording to the invention bind in a considerably inferior manner thanthe unesterified glucocorticoid framework to the glucocorticoid receptorof the rat thymus. In this respect, they meet the condition of aprodrug.

                  TABLE 1                                                         ______________________________________                                        Competition Factors in the Cytosolic Rat Thymus-                              Glucocorticoid Receptor                                                                                    Competition                                      Corticoid          Example   Factor [KF]                                      ______________________________________                                        6α-Methylprednisolone-17-                                                                            0.8                                              propionate                                                                    Boc-Val-O-MPP       1         17                                              Boc-Ala-Ala-Pro-Val-O-MPP                                                                         3        5.6                                              Fmoc-Ala-Ala-Pro-Val-O-MPP                                                                        4        100                                              Ac-Ala-Ala-Pro-Val-O-MPP                                                                          7         20                                              Bz-Ala-Ala-Pro-Val-O-MPP                                                                          8         24                                              Valeroyl-Ala-Ala-Pro-Val-O-                                                                       9         7                                               MPP                                                                           6α-Methylprednisolone  0.9                                              Boc-Val-O-MP       19        144                                              Boc-Ala-Ala-Pro-Val-O-MP                                                                         21         56                                              Fmoc-Ala-Ala-Pro-Val-O-MP                                                                        22        220                                              Hydrocortisone                4                                               Boc-Val-O-HC       27        >1000                                            Fmoc-Ala-Ala-Pro-Val-O-HC                                                                        29        k.k. [n.c.]                                      Boc-Ala-Ala-Pro-Val-O-FC                                                                         32         5                                               Boc-Ala-Ala-Pro-Val-O-TCA                                                                        44         24                                              Bz-Ala-Ala-Pro-Val-O-TCA                                                                         47         10                                              ______________________________________                                         (k.k. = no competition)                                                  

2. Cleavage in the Homogenate of Normal Rat Skin

The homogenate of the skin of male rats (0.1 mol/l of phosphate buffer,pH=7.4) is centrifuged for 20 minutes at 10,000 g. The supernatant isadjusted in phosphate buffer to a protein concentration of 3 mg/ml. Thetest substances in 20 μl of ethanol are added to 1 ml of thishomogenate. The final concentration of the corticoid derivatives isapproximately 100 μmol/l. The incubation takes place for differentperiods at 37° C. At the end of incubation, the samples are extractedthree times with 3 ml of chloroform. The combined extracts areevaporated to dryness under nitrogen and taken up with 100 μl ofethanol. The ethanolic solutions are mixed with the same volume of waterand the saponification products are separated by HPLC (RP 18,acetonitrile/water-gradient, detection 240 nm). The relative hydrolysisrate is indicated in percent of the hydrolysis rate of the6α-methylprednisolone-17-propionate-21-acetate.

The oligopeptide esters have proven to be less hydrolysis-sensitive thansimple carboxylic acid esters (Table 2).

                  TABLE 2                                                         ______________________________________                                        Unspecific Cleavage of Corticoid-21-Oligopeptide                              Esters by Esterases of Rat Skin                                                                             Relative                                                                      Hydrolysis                                      Corticoid            Example  Rate [%]                                        ______________________________________                                        6α-Methylprednisolone-17-                                                                             100                                             propionate-21-acetate                                                         Boc-Ala-Ala-Pro-Val-O-MPP                                                                           3       50                                              Fmoc-Ala-Ala-Pro-Val-O-MPP                                                                          4       4                                               DPAc-Ala-Ala-Pro-Val-O-MPP                                                                         10       6                                               DPC-Ala-Ala-Pro-Val-O-MPP                                                                          11       14                                              Naphthoyl-Ala-Ala-Pro-Val-O-MPP                                                                    12       4                                               Cinnamoyl-Ala-Ala-Pro-Val-O-MPP                                                                    13       5                                               Cbs-Ala-Ala-Pro-Val-O-MPP                                                                          14       6                                               Cbp-Ala-Ala-Pro-Val-O-MPP                                                                          16       3                                               Pht-Gly-Ala-Ala-Pro-Val-O-MPP                                                                      17       49                                              Fmoc-Ala-Ala-Pro-Val-O-MP                                                                          22       6                                               Boc-Ala-Ala-Pro-Val-O-BMV                                                                          38       0                                               Fmoc-Ala-Ala-Pro-Val-O-BMV                                                                         39       0                                               ______________________________________                                    

3. Cleavage of Glucocorticoid-21-Oligopeptide Esters by LeukocyteElastase

Polymorphonuclear granulocytes are isolated from donor blood. The cellsare lysed in a density of 10⁶ cells per ml. 1 ml each of the lysate ismixed with 20 μl of ethanolic corticoid solution (5 mmol/l) After 1 hourof incubation at 37° C., the samples are extracted three times with 3 mlof chloroform and worked up as described above for HPLC analysis. Thecleavage of the prodrugs according to the invention can be inhibitedunder these test conditions by the addition of the specific elastaseinhibitor MeO-succinyl-Ala-Ala-Pro-Val-chloromethylketone to 90%.Furthermore, the compounds are also hydrolyzed by pure humansputum-elastase (elastin products). These findings suggest a specificcleavage of the prodrugs by elastase.

                  TABLE 3                                                         ______________________________________                                        Cleavage of Glucocorticoid-21-esters in the Lysate of                         Human Neutrophilic Granulocytes                                                                             Relative                                                                      Hydrolysis                                      Corticoid            Example  Rate [%]                                        ______________________________________                                        Boc-Ala-Ala-Pro-Val-O-MPP                                                                           3       160-190                                         Fmoc-Ala-Ala-Pro-Val-O-MPP                                                                          4       100                                             Z-Ala-Ala-Pro-Val-O-MPP                                                                             5       160-230                                         Fmoc-Aib-Ala-Ala-Pro-Val-O-MPP                                                                     18       130                                             Fmoc-Ala-Ala-Pro-Val-O-MP                                                                          22       370                                             Boc-Ala-Ala-Pro-Val-O-FC                                                                           32       380                                             Fmoc-Ala-Ala-Pro-Val-O-BMV                                                                         39        42                                             ______________________________________                                         Fmoc-Ala-Ala-Pro-Val-O-MPP is used as reference.                         

4. Local Antiinflammatory Action

The antiinflammatory activity of the corticoid-21-oligopeptide esters isperformed in a modified rat ear test according to Tonelli. Themodifications are based on changes of irritant solution (4% croton oil,10% DMSO in ethanol), the type of administration (application of thesolution with pipette) and the evaluation of the test 16 hours afteradministration of the irritant solution. At this time, both theinhibition of the edema by measuring the ear weight and the inhibitionof the granulocytic infiltrate by measuring the granulocyte markermyeloperoxidase and leukocyte-elastase in the ear homogenate can bedetermined. The group size was 6 animals in each case. As can be seenfrom Table 4, the antiinflammatory activity of thecorticoid-21-oligopeptide esters after topical application is comparableto the activity of the homologous corticoid-21-acetates.

                  TABLE 4                                                         ______________________________________                                        Antiinflammatory Action of Glucocorticoid-21-Esters                           after Topical Application                                                     (Rat Ear Test)                                                                                       Edema      Infiltration                                Corticoid  Concentration                                                                             [% inhibition]                                                                           [% inhibition]                              ______________________________________                                        6α-Methylpred-                                                                     0.3         67*        78*                                         nisolone-17-                                                                             0.03        62*        45*                                         propionate-21-                                                                           0.003       44*        12                                          acetate                                                                       Boc-Ala-Ala-                                                                             0.3         81*        77*                                         Pro-Val-O-MPP                                                                            0.03        51*        43*                                         (Example 3)                                                                              0.003       17         11                                          Fmoc-Ala-Ala-                                                                            0.3         78*        66*                                         Pro-Val-O-MPP                                                                            0.03        48*        42*                                         (Example 4)                                                                              0.003       30*        16*                                         ______________________________________                                         *Significant inhibition (p < 0.05) relative to positive control               (application of croton oil without corticoid)                            

We claim:
 1. A glucocorticoid of general formula I

    R--Val--O--GC (I),

in which O-GC is the radical of a 21-hydroxycorticoid that has an antiinflammatory action, Val represents a valine radical in the 21-position of the corticoid and R means a hydrogen atom or a hydrocarbon radical with up to 32 carbon atoms that is optionally substituted by hydroxy groups, amino groups, oxo groups and/or halogen atoms and/or interrupted by oxygen atoms, SO₂ groups and/or NH groups a salt thereof.
 2. A pharmaceutical preparation comprising a glucocorticoid according to claim 1 and a pharmaceutically acceptable carrier.
 3. A glucocorticoid of general formula II ##STR2## in which R¹ =H, CH=O, (C=O)R", (C=O)OR" or SO₂ R"X¹ -X³ =independently of one another, alanine, proline or valine, A-B=CH₂ --CH₂ or CH=CH R⁶ =H, F, Cl, Me, R⁹ =H, F, Cl, R¹⁶ =H, Me, OH, R¹⁷ =H, OH, O(C=O)R'" or R¹⁶, R¹⁷ =alkylidenedioxy, in which R" represents a hydrocarbon radical that contains C₁ -C₁₈ and R'" represents a straight-chain a branched-chain C₁ -C₁₀ alkyl, aryl, alkylaryl or C₁ -C₃ alkoxy radical and the alkylidene radical is derived from an aliphatic aldehyde that contains 1-6 carbon atoms, a ketone that contains 3-6 carbon atoms or cyclic ketone or benzaldehyde that contains 5-6 carbon atoms or a salt therof.
 4. A pharmaceutical preparation comprising a glucocorticoid according to claim 3 and a pharmaceutically acceptable carrier.
 5. A glucocorticoid of general formula I

    R--Val--O--GC (I),

in which O--GC is the radical of a 21-hydroxycorticoid that has an antiinflammatory action, Val represents a valine radical in the 21-position of the corticoid and R means a hydrogen atom or a hydrocarbon radical with up to 32 carbon atoms that is optionally substituted by hydroxy groups, amino groups, oxo groups and/or halogen atoms and/or interrupted by oxygen atoms, SO₂ groups and/or NH groups; or general formula II ##STR3## in which R¹ =H, CH=O, (C=O)R" or SO₂ R", X¹ and X² =alanine, X³ =proline or valine, A--B=CH₂ --CH₂ or CH=CH, R⁶ =H, F, Cl, Me, R⁹ =H, F, Cl, R¹⁶ =H, Me, OH, R¹⁷ =H, OH, O(C=O)R'" or R¹⁶, R¹⁷ =alkylidenedioxy, in which R" represents a hydrocarbon radical that contains C₁ -C₁₈ and R'" represents a straight-chain or branched-chain C₁ -C₁₀ alkyl, aryl, alkylaryl or C₁ -C₃ alkoxy radical and the alkylidene radical is derived from an aliphatic aldehyde that contains 1-6 carbon atoms, a ketone that contains 3-6 carbon atoms or cyclic ketone or benzaldehyde that contains 5-6 carbon atoms or a salt thereof.
 6. A method for treating inflammatory conditions, wherein a glucocorticoid according to claim 5 is administered to a patient in need thereof. 